Tuberculosis has re-emerged as a threat to global health. Increasing incidences and increases in the numbers of drug-resistance are sounding alarms for a disease that was once thought cured. Infection with drug- sensitive strains of Mycobacterium tuberculosis can be effectively cured with a combination of isoniazid (INH), rifampicin, and pyrazinamide. However, the emergence of multiple drug resistant strains of M. tuberculosis has resulted in fatal outbreaks in the United States. Although INH is one of the most widely used anti-tuberculosis drugs for both therapy and prophylaxis, its precise target of action on Mycobacterium tuberculosis was unknown. We have discovered a novel gene, named inhA, in M. tuberculosis and all mycobacterial species examined that encodes a target for both isoniazid and ethionamide. Two different genetic mechanisms have been determined that mediate isoniazid and ethionamide resistance: i) point mutations that cause amino acid substitutions in the structural gene or ii) overexpression of the structural gene. Resistance to isoniazid can also be mediated by mutations in the catalase peroxidate gene (katG) of M. tuberculosis. The focus of this Project is to use a molecular genetic approach to elucidate the relationships between katG and inhA. Coupled with the biochemical and structural analysis of this proposal, these strategies provide a comprehensive approach for elucidating the precise mechanisms of ethionamide and isoniazid actions.